We are interested in the transcriptional control of T cell development and function, and specifically in the gene expression programs that control the choice by intrathymic T cell precursors of the CD4 or CD8 lineage, and perpetuate lineage differentiation in post-thymic T cells. T cells are essential to the function of the immune system. Most T cells (generally referred to as 'conventional'T cells) recognize peptide antigens presented by class I (MHC-I) or class II (MHC-II) classical Major Histocompatibility Complex molecules, and express either of two surface glycoproteins (called coreceptors) that contribute to antigen recognition: CD4, which binds MHC-II, or CD8, which binds MHC-I. Coreceptor expression on mature T cells is mutually exclusive and strongly correlates with both MHC specificity and functional differentiation. That is, the general rule is that MHC I-specific T cells are CD4-CD8+ and cytotoxic (CD8 cells), whereas MHC II-specific T cells are CD4+CD8- and helper or regulatory (CD4 cells). This double correspondence is essential to the proper function of the immune system and is established in the thymus, where CD4 and CD8 T cells emerge as separate lineages from precursors expressing both CD4 and CD8 (double positive, DP). Deciphering the transcriptional regulatory networks that decide and maintain CD4-CD8 lineage differentiation is the target of our current research. During the period covered by this report, our work has focused on two specific aspects of this problem, First, based on our earlier finding that the two zinc finger transcription factors Gata3 and Thpok serve distinct functions in the differentiation of conventional CD4 cells (Wang et al, 2008, Nat. Immunol.), we have examined whether and how the interplay of these factors affected the development of other T lineages. Recent advances have put emphasis on non-conventional T cells that typically recognize non-peptide antigens associated with MHC or with MHC-like molecules. One such subset, called invariant iNK T (iNK T) cells because fo their limited TCR repertoire diversity, recognizes lipid antigens associated with the surface molecule CD1d. These cells, that can be either CD4+CD8- or CD4-CD8-, are found in high numbers at sites of effector responses such as the liver or the gut. We have evaluated the respective roles of Gata3 and Thpok in the development of iNK T cells. We found that Thpok is expressed by all iNK T cells, regardless of whether they are CD4+CD8- or CD4-CD8-, and that Gata3 is required for appropriate Thpok expression in each subset. The function of Thpok in iNK T cell differentiation is more complex. Similar to conventional T cells, Thpok is not required for the development of iNK T cells, but is necessary for their expression of CD4 and their repression of CD8. However, unlike in conventional T cells, Thpok does not appear to repress expression of genes associated with the cytotoxic effector program. To the contrary, we found that Thpok was necessary for the expression of Granzyme B, a prototypical cytotoxic gene, by NK T cells, whether they express CD4 or not. Thus, the sets of functions Thpok serves in distinct T cell subset subsets are only partially overlapping. We are currently exploring the mechanistic bases for this difference. The second area we have explored relates to the expression of Runx3, a transcription factor important for CD8 T cell development. Work by several groups (notably those of Dan Littman, Yoram Groner and Ichiro Taniuchi) has emphasized the role of Runx transcription factors in this process. Specifically, Runx3 appears essential to CD8-lineage differentiation, in a manner partly redundant with Runx1, by promoting the cessation of Cd4 expression. We and others had also found that Runx3 promotes the expression of genes characteristic of the cytotoxic program. While Runx3 expression is characteristic of CD8-lineage precursors in the thymus, how it is controled has remained unclear. In light of previous studies that had identified another transcription factor, Ets1, as important for CD8 cell differentiation, we examined how Ets1 was connected to Runx3. In fact, the consequences of Ets1 disruption in CD8-differentiating cells were strikingly similar to those of Runx3 disruption, dominated by an impaired termination of Cd4 gene expression. Indeed, we found that Ets1-deficient CD8-lineage thymocytes had reduced expression of Runx3, and that Ets1 was recruited to at least three binding sites located within the Runx3 locus. To verify that such reduced expression of Runx3 was responsible for the impaired CD8-differentiation of Ets1-deficient thymocytes, we introduced into Ets1-deficient mice a Runx3 transgene expressed throughout T cell development. We found that enforced expression of Runx3 largely corrected the defective Cd4 repression in Ets1-deficient thymocytes. Altogether, these findings identify Ets1 as a novel node in the circuitry that promotes CD8 lineage differentiation of MHC I-restricted thymocytes and show that it acts at least in part by promoting Runx3 expression.